U.S. patent application number 15/117613 was filed with the patent office on 2016-12-01 for method for producing phenyl propane-based compound using microorganisms.
This patent application is currently assigned to JAPAN AGENCY FOR MARINE-EARTH SCIENCE AND TECHNOLOGY. The applicant listed for this patent is JAPAN AGENCY FOR MARINE-EARTH SCIENCE AND TECHNOLOGY. Invention is credited to Ryoichi HASEGAWA, Yuji HATADA, Yukari OHTA.
Application Number | 20160348135 15/117613 |
Document ID | / |
Family ID | 53778073 |
Filed Date | 2016-12-01 |
United States Patent
Application |
20160348135 |
Kind Code |
A1 |
OHTA; Yukari ; et
al. |
December 1, 2016 |
METHOD FOR PRODUCING PHENYL PROPANE-BASED COMPOUND USING
MICROORGANISMS
Abstract
The object of the invention is to provide a method which
specifically and efficiently produces a compound having a phenol
propane structure from natural biomass containing lignins by
causing microorganisms to act on biomass. The object is achieved by
a method for producing a phenyl propane-based compound comprising a
step of producing a phenyl propane-based compound by causing
microorganisms of the genus Novosphingobium to act on biomass
containing lignins and/or lignin-related substances.
Inventors: |
OHTA; Yukari; (Yokosuka-shi,
JP) ; HATADA; Yuji; (Yokosuka-shi, JP) ;
HASEGAWA; Ryoichi; (Yokosuka-shi, JP) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
JAPAN AGENCY FOR MARINE-EARTH SCIENCE AND TECHNOLOGY |
Yokosuka-shi |
|
JP |
|
|
Assignee: |
JAPAN AGENCY FOR MARINE-EARTH
SCIENCE AND TECHNOLOGY
Yokosuka-shi
JP
|
Family ID: |
53778073 |
Appl. No.: |
15/117613 |
Filed: |
February 9, 2015 |
PCT Filed: |
February 9, 2015 |
PCT NO: |
PCT/JP2015/053547 |
371 Date: |
August 9, 2016 |
Current U.S.
Class: |
1/1 |
Current CPC
Class: |
C12P 7/22 20130101; C12R
1/01 20130101; C12P 7/24 20130101; C12N 1/20 20130101 |
International
Class: |
C12P 7/22 20060101
C12P007/22; C12R 1/01 20060101 C12R001/01; C12N 1/20 20060101
C12N001/20 |
Foreign Application Data
Date |
Code |
Application Number |
Feb 10, 2014 |
JP |
2014-023839 |
Claims
1. A method for producing a phenyl propane-based compound
comprising a step of producing a phenyl propane-based compound by
causing microorganisms of the genus Novosphingobium to act on
biomass containing lignins and/or lignin-related substances.
2. The method according to claim 1, wherein the phenyl
propane-based compound is at least one selected from a group of
3-hydroxy-1-(4-hydroxy-3-methoxyphenyl)-1-propanone,
3-hydroxy-1-(4-hydroxy-3,5-dimethoxyphenyl) -1-propanone and
3-hydroxy-1-(4-hydroxyphenyl)-1-propanone.
3. The method according to claim 1, wherein the microorganisms of
the genus Novosphingobium are those of Novosphingobium species
MBES04 (accession number: NITE P-01797).
4. Novosphingobium species MBES04 (accession number: NITE P-01797).
Description
CROSS REFERENCE TO RELATED APPLICATIONS
[0001] This invention claims the benefit of priority of Japanese
Patent Application No. 2014-023839, filed on Feb. 10, 2014, which
is incorporated by reference herein in its entirety.
TECHNICAL FIELD
[0002] This invention relates to a method for specifically
producing a compound having a phenyl propane structure from biomass
containing lignins and lignin-related substances by using
microorganisms and also to microorganisms to be used for the
method.
BACKGROUND ART
[0003] Lignins are amorphous polymeric substances existing in
plants as ingredients of vascular bundle cell walls. Lignins are
formed as a result of complex condensation reactions of phenyl
propane-based constituent units and show a remarkable chemical
structural characteristic of containing methoxy groups. Lignins
take a role of causing lignified plant cells to mutually
agglutinate, thereby strengthening plant tissues. Lignins are
contained by 18 to 36% in woods and by 15 to 25% in herbaceous
plants. Thus, various attempts have been and being made to degrade
lignins and obtain useful compounds therefrom for the purpose of
effectively exploiting wood resources.
[0004] Meanwhile, known phenyl propane-based compounds include
coumaric acid, cinnamic acid, caffeic acid (3,4-dihydorxy-cinnamic
acid), eugenol, anethol, coniferyl alcohol, sinapyl alcohol and
ferulic acid. In industrial fields, phenyl propane-based compounds
are useful because such compounds can be used for medicines,
functional foods and synthetic intermediates of various chemical
products such as perfumes, spices, essential oils, fungicides,
anesthetics and antioxidant agents.
[0005] For example, methods for non-specifically degrading lignins
contained in lignocellulose substances of woods, rice straws and so
on down to low molecules by using physiochemical techniques such as
gasifying techniques involving high-temperature high-pressure
processing (see Patent Literatures 1 and 2 listed below, which are
incorporated by reference herein in their entirety) and pressurized
hot water treatment techniques (see Patent Literature 3 listed
below, which is incorporated by reference herein in its entirety)
are known. However, with any of the above listed techniques, it is
very difficult to produce a specific compound from lignins. In
other words, when any of the above listed techniques is employed,
phenyl propane-based compounds having three carbon atoms in each
carbon side chain that is directly bonded to a benzene ring
skeleton, which compounds operate as unit structures of lignins,
are further degraded to lower molecules. To be more accurate,
phenyl propane-based compounds are non-specifically transformed
into phenols such as guaiacols, sylingols and so on where one or
more than one carbon side chains having no carbon atoms are
directly bonded to a benzene ring skeleton and also into phenyl
methane compounds such as vanillin, syringaldehyde and so on where
one or more than one carbon side chains having one carbon atoms are
directly bonded to a benzene ring skeleton. Additionally,
degradation products of lignins are mixtures of a variety of
components and hence it is highly difficult to obtain only phenyl
propane-based compounds therefrom. In other words, it is not
possible to specifically produce phenyl propane-based compounds
with any of the above identified techniques. Furthermore, if any of
the above listed physiochemical methods is adopted and put to
practical use, it requires a vast amount of energy and special
apparatus.
[0006] In view of the above-identified problems, attempts are being
made to obtain degradation products of lignins by biologically
processing lignins. For example, a method of degrading lignins by
inoculating white-rot fungus into lignocellulose substances and
incubating them there is known (see Patent Literature 4 listed
below, which is incorporated by reference herein in its
entirety).
[0007] Meanwhile, since it is known that .beta.-aryl ether type
bonds account for about 50% of the chemical bonds existing in
natural lignins, if .beta.-aryl ether bonds can be cleaved or not
is highly significant from the viewpoint of degrading natural
lignins. Known microorganisms that produce enzymes capable of
specifically cleaving .beta.-aryl ether type bonds include
microorganisms of the genus Sphingobium (see Patent Literature 5
and Non-Patent Literature 1 listed below, which are incorporated by
reference herein in their entirety; note, however, microorganisms
of that genus are described as those the genus Pseudomonas in
Non-Patent Literature 1), microorganisms of the genus Brevundimonas
(see Patent Literature 6, which is incorporated by reference herein
in its entirety) and microorganisms of the genus Pseudomonas (see
Non-Patent Literature 2 listed below, which is incorporated by
reference herein in its entirety).
[0008] Patent Literatures 4 through 6 and Non-Patent Literatures 1
and 2 describe microorganisms of the genus Sphingobium,
microorganisms of the genus Brevundimonas and microorganisms of the
genus Pseudomonas as well as methods of producing phenyl
propane-based compounds by cleaving .beta.-aryl ether type bonds of
guaiacylglycerol-.beta.-guaiacyl ether or
3-hydroxy-2-(2-methoxyphenoxy)-1-(3-methoxy-4-hydroxypheny
1)-1-propanone, which are model compounds of lignins, by means of
enzymes capable of cleaving .beta.-aryl ether type bonds that are
produced by microorganisms of any of the above listed genera.
PRIOR ART LITERATURES
Patent Literatures
[0009] Patent Literature 1: JP 2012-140346 A
[0010] Patent Literature 2: JP 2012-50924 A
[0011] Patent Literature 3: JP 2010-239913 A
[0012] Patent Literature 4: JP 1975-46903 A
[0013] Patent Literature 5: JP 1993-336976 A
[0014] Patent Literature 6: JP 2002-34557 A
Non-Patent Literatures
[0015] Non-Patent Literature 1: FEBS Lett. 249 (2), 1989, pp.
348-352
[0016] Non-Patent Literature 2: Mokuzai Gakkaishi, Vol. 31 (11),
1985, pp. 956-958
[0017] Non-Patent Literature 3: Biosci Biotechnol Biochem. 2011; 75
(12):2404-7
DISCLOSURE OF THE INVENTION
Problem to be solved by the Invention
[0018] According to the description of Patent Literature 4, the
method described in the literature can provide possibility of
degrading lignins by causing microorganisms to act on the lignins
to be degraded. However, when white-rot fungi and ligninase that
white-rot fungi produce are caused to act on lignocellulose
substances, not only the products lack structural uniformity but
also polymerization reactions mainly of the products progress
because the specificity of the lignin degrading reaction caused by
the microorganisms is very low. Therefore, the method described in
Patent Literature 4 has a problem that the reaction products lack
usefulness as industrial raw materials.
[0019] The methods described in Patent Literatures 5 and 6 and
Non-Patent Literatures 1 through 3 utilize microorganisms and
enzymes produced by microorganisms of the type under consideration
to produce phenyl propane-based compounds from model compounds of
lignin. However, these literatures do not describe any fact that
phenyl propane-based compounds are obtained from natural biomass by
causing microorganisms and enzymes to act on model compounds of
lignin. In particular, it is not possible to produce phenyl
propane-based compounds only by using the enzymes described in
Patent Literature 5. It is not possible either to produce phenyl
propane-based compounds by using the microorganisms described in
Non-Patent Literature 1 because those microorganisms completely
break down guaiacylglycerol-.beta.-guaiacyl ether down to CO2 for
anabolism.
[0020] While microorganisms described in Non-Patent Literature 2
are capable of metabolizing guaiacylglycerol-.beta.-guaiacyl ether,
the yield of the obtained product is very low because the produced
phenyl propane-based compounds are further metabolized and
transformed into different compounds.
[0021] Therefore, the problem to be solved by the invention is to
provide a method which, if compared with the methods described in
Patent Literature 5 and 6 and Non-Patent Literature 1 and 2, more
specifically and efficiently produces a compound having a phenyl
propane structure from natural biomass containing lignins by
causing microorganisms to act on the biomass.
Means for Solving the Problem
[0022] The inventors of the present invention have paid intensive
research efforts to solve the above identified problem, conducting
operations of screening microorganisms obtained from sunken woods
in deep seas and capable of degrading lignin-related compounds. As
a result, the inventors of the present invention succeeded in
isolating a novel strain of microorganisms that degrade
lignin-related compounds from more than 1,000 strains of
microorganisms. Furthermore, not only microorganisms of the strain
can produce phenyl propane-based compounds from model compounds of
lignins but also they can specifically produce phenyl propane-based
compounds, using natural lignin-containing biomass as
substrates.
[0023] Additionally, the inventors of the present invention found
that microorganisms of the strain can efficiently degrade model
compounds of lignins and produce phenyl propane-based compounds at
a high yield. The present invention is based on these successful
researches and findings.
[0024] Thus, the present invention provides a method for producing
a phenyl propane-based compound comprising a step of producing a
phenyl propane-based compound by causing microorganisms of the
genus Novosphingobium to act on biomass containing lignins and/or
lignin-related substances.
[0025] Preferably, in the production method of the present
invention, the phenyl propane-based compound is at least one
selected from a group of
3-hydroxy-1-(4-hydroxy-3-methoxyphenyl)-1-propanone,
3-hydroxy-1-(4-hydroxy-3,5-dimethoxyphenyl) -1-propanone and
3-hydroxy-1-(4-hydroxyphenyl)-1-propanone.
[0026] Preferably, in the production method of the present
invention, microorganisms of the genus Novosphingobium are those of
Novosphingobium species MBES04 (accession number: NITE
P-01797).
[0027] In another aspect of the present invention, there is
provided Novosphingobium species MBES04 (accession number: NITE
P-01797).
Advantages of the Invention
[0028] Thus, with the production method and microorganisms
according to the present invention, it is possible to specifically
and efficiently produce a compound having a phenyl propane
structure from biomass containing lignins and lignin-related
substances and originating from natural materials such as
agricultural wastes and woods.
BRIEF DESCRIPTION OF THE DRAWINGS
[0029] FIG. 1 is a graph illustrating the change with time of the
quantity of a substrate and also the quantity of the reaction
product obtained by causing guaiacylglycerol-.beta.-guaiacyl ether
to act on MBES04 strain. In the graph, GGGE represents
guaiacylglycerol-.beta.-guaiacyl ether and GPGE represents
1-(4-hydroxy-3-methoxyphenyl)-3-hydroxy-2-(2-methoxyphenoxy)propane-1-on,
while GHP represents
3-hydroxy-1-(4-hydroxy-3-methoxyphenyl)-1-propanone and Guaiacol
simply means guaiacol.
[0030] FIG. 2 is a schematic illustration of the chromatogram of
the culture supernatant obtained by causing
guaiacylglycerol-.beta.-guaiacyl ether to act on MBES04 strain. In
the graph, the vertical axis (UV 270 nm Intensity) indicates the
intensity of UV rays at the wavelength of 270 nm.
BEST MODE FOR CARRYING OUT THE INVENTION
[0031] Now, the present invention will be described in greater
detail below.
[0032] The production method of the present invention is a method
of producing a phenyl propane-based compound comprising a step of
obtaining a phenyl propane-based compound by causing microorganisms
of the genus Novosphingobium to act on biomass containing lignins
and/or lignin-related substances.
[0033] With the production method of the present invention, phenyl
propane-based compounds such as
3-hydroxy-1-(4-hydroxy-3-methoxyphenyl)-1-propanone,
3-hydroxy-1-(4-hydroxy-3,5-dimethoxyphenyl)-1-propanone and
3-hydroxy-1-(4-hydroxyphenyl)-1-propanone are produced as
microorganisms of the genus Novosphingobium are caused to exert
metabolic effects on lignins and lignin-related substances in
biomass.
[0034] There are no particular limitations to the phenyl
propane-based compound that can be obtained by the above defined
production method of the present invention so long as it is a
compound having a phenyl propane structure. For example, a phenyl
propane-based compound that can be obtained by the above defined
production method of the present invention may be a compound
expressed by general formula (A) shown below:
##STR00001##
(where R represents 1 or more than 2 alkyl groups or alkoxy groups
having 1 to 5 carbon atoms or hydrogen atoms) that has a carbonyl
group at 1-position and hydroxyl groups respectively at 3-position
and at 4-position of a phenyl group. More specifically, the
compound is 3-hydroxy-1-(4-hydroxy-3-methoxyphenyl) -1-propanone,
3-hydroxy-1-(4-hydroxy-3,5-dimethoxyphenyl)-1-propanone or
3-hydroxy-1-(4-hydroxyphenyl)-1-propanone and, more preferably, it
is 3-hydroxy-1-(4-hydroxy-3-methoxyphenyl)-1-propanone.
[0035] Of the above listed compounds, for example,
3-hydroxy-1-(4-hydroxy-3-methoxyphenyl)-1-propanone has a structure
expressed by formula (I) shown below.
##STR00002##
[0036] 3-hydroxy-1-(4-hydroxy-3-methoxyphenyl)-1-propanone can
qualitatively and quantitatively be analyzed, for example, by means
of reverse phase HPLC. The conditions to be met for reverse phase
HPLC include that an Octa Decyl Silyl group-modified silica gel
column (ODS column) is to be employed along with eluent A (2 mM
ammonium acetate, 0.05% V/V formic acid) and eluent B (100% V/V
methanol), that a column temperature of 40.degree. C. and a flow
rate of 1.2 ml/min are to be set and that a mixture solution of
eluent A 90% V/V and eluent B 10% V/V is to be fed for a minute and
subsequently eluent B is to be fed at a gradient of 10% V/V to 95%
V/V for seven minutes. As the above conditions are satisfied,
3-hydroxy-1-(4-hydroxy-3-methoxyphenyl)-1-propanone can be detected
with a peak of retention time of around 4.5 minutes by using a UV
detector (270 nm). It can be quantified by means of a calibration
curve method, an internal standard method or the like provided that
standard 3-hydroxy-1-(4-hydroxy-3-methoxyphenyl)-1-propanone is
employed.
[0037] The above defined production method of the present invention
employs biomass containing lignins and/or lignin-related
substances. While lignins to be used as starting material are not
subjected to any particular limitations so long as they are known
to those who are skilled in the art, they are typically found in
vascular bundles of plants and known to have a complicatedly
polymerized dendritic structure where 3 types of phenylpropanoid
that are expressed respectively by chemical formulas (B) through
(D) shown below operate as unit constituents.
##STR00003##
[0038] Lignin-related substances to be used for the purpose of the
present invention are not subjected to any particular limitations
so long as they are substances derived from lignins. As far as this
specification of the present invention is concerned, lignin-related
substances include substances that are regarded as model compounds
of lignin such as guaiacylglycerol-.beta.-guaiacyl ether in
addition to degradation products of lignins and substances that can
be obtained by processing lignins. As far as this specification is
concerned, biomass is not subjected to any particular limitations
so long as it contains lignins and/or lignin-related substances.
For example, biomass includes natural products such as grasses and
trees, substances that can be obtained by processing grasses and
trees and agricultural wastes.
[0039] Biomass containing lignins and lignin-related substances
(which may also be referred to as lignin-containing biomass
hereinafter) can take any of the forms of, for example, solid,
suspension and liquid and the forms that biomass can take vary
depending on if it is preprocessed or not. For example,
lignin-containing biomass can take the form of suspension obtained
by crushing the biomass and adding liquid to the crushed
biomass.
[0040] Lignin-containing biomass may be extracted lignins. Examples
of extracted lignins include extracted liquid lignins obtained by
powdering lignin-containing biomass, preparing a suspension by
causing the powdered lignin-containing biomass to be suspended in a
solvent that is suited for extraction of lignins so as to produce a
suspension of between 0.1% W/V and 50% W/V, preferably between 1%
W/V and 20% W/V, subjecting the suspension to an extraction process
at temperatures between 10.degree. C. and 150.degree. C.,
preferably between 20.degree. C. and 130.degree. C., more
preferably between 20.degree. C. and 80.degree. C., for a period
between several hours and several days, preferably between an hour
and 6 days, and subsequently removing the solid contents from the
extraction-processed solution and extracted solid lignins obtained
by removing the solvent from the extracted liquid lignins and
drying the evaporation residue.
[0041] Solvents that can be used for the purpose of the present
invention and are suited for extraction of lignins are not
subjected to any particular limiations. Examples of such solvents
include water, dioxane, low molecular weight alcohols such as
methanol and isopropanol, dimethyl formaldehyde and so on, of which
water and dioxane are preferable.
[0042] With the above defined production method of the present
invention, enzymes derived from microorganisms that belong to the
genus Novosphingobium are employed in order to obtain phenyl
propane-based compounds from lignin-containing biomass. Any
microorganisms belonging to the genus Novosphingobium, which is
also referred to as the genus Sphingomonas, may be used for the
purpose of the present invention without limitations provided that
they are gram-negative bacilli having a size of 0.3 to 0.8.times.2
.mu.m to 3 .mu.m. Examples of such microorganisms include those
belonging to the genus Novosphingobium that are known to degrade
various aromatic compounds. Specific preferable examples of
microorganisms of the genus Novosphingobium that can be used for
the production method of the present invention include
Novosphingobium species MBES04 (to be also referred to as MBES04
strain hereinafter).
[0043] The mycological properties, the physiological properties and
the utilization potential as substrate of MBES04 strain will be
described hereinafter in Examples 3 and 4. The base sequence of the
DNA that encodes 16S ribosome RNA of MBES04 strain is described in
the sequence listing with the sequence number 3 (accession number:
AB733576). In view of these descriptions, therefore, microorganisms
of the genus Novosphingobium that have the mycological properties,
the physiological properties and the utilization potential as
substrate or the base sequence of MBES04 strain can be regarded as
those that correspond to or are equivalent to microorganisms of
MBES04 strain. Particularly, microorganisms of the genus
Novosphingobium that show agreement with the enzyme activity
described in Table 1 below by not less than 90%, preferably not
less than 95%, more preferably not less than 99%, agreement with
the utilization potential as substrate described in Table 2 shown
below by not less than 90%, preferably not less than 95%, more
preferably not less than 99% and/or identity with the base sequence
described in the sequence listing with the sequence number 3 by not
less than 98%, preferably not less than 99%, more preferably not
less than 99.5% can be regarded substantially as those of MBES04
strain. As far as this specification is concerned, the expression
of "identity" of sequences means strict identity of two sequences
that is observed when the two sequences are aligned with each other
and does not mean similarity between the two sequences.
[0044] Likewise, microorganisms of the genus Novosphingobium to be
used for the production method of the present invention can be
isolated from woods or the like in the realm of nature where, for
example, corrosion and rottenness are observed, on the basis of the
mycological properties, the physiological properties and the
utilization potential as substrate and/or the base sequence of
MBES04 strain. While there are no particular limitations to the
method for isolating microorganisms of MBES04 strain, purely
cultured microorganisms of the strain can be obtained, for example,
by means of a streak culture method and a limiting dilution
method.
[0045] Phenyl propane-based compounds can be obtained by causing
microorganisms of the genus Novosphingobium to act on
lignin-containing biomass. Note that the expression of "causing
microorganisms of the genus Novosphingobium to act on" as used
herein means causing microorganisms of the genus Novosphingobium to
exert the potential ability they have of transforming lignins and
lignin-related substances into phenyl propane-based compounds.
[0046] There are no particular limitations to the conditions of a
system (to be referred to as action system hereinafter) for causing
lignin-containing biomass to act on microorganisms of the genus
Novosphingobium so long microorganisms of the genus Novosphingobium
can survive under the conditions. For example, they are the
conditions under which microorganisms of the genus Novosphingobium
can be cultured. When microorganisms of the genus Novosphingobium
are those of MBES04 strain, for example, the conditions under which
such microorganisms can be cultured include temperatures between 10
and 45.degree. C., preferably between 15 and 37.degree. C., pH
values between 5.5 and 8.5, preferably between 6 and 8 and NaCl
concentrations between 0 and 6% W/V, preferably between 0 and 3%
W/V.
[0047] Additionally, culture medium ingredients may be allowed to
exist in such an action system. There are no particular limitations
to culture medium ingredients that can be used for the purpose of
the present invention so long as they are normally known culture
medium ingredients for microorganisms. Such culture medium
ingredients typically include carbon sources, nitrogen sources,
inorganic substances and micronutrients that may be required for
the microorganisms strain to be used. These ingredients may be
either natural substances or synthesized substances.
[0048] There are no particular limitations to carbon sources to be
used for the purpose of the present invention so long as they have
utilization potential for microorganisms of the genus
Novosphingobium. Examples of carbon sources include carbohydrates
such as glucose, lactose, maltose, cellobiose, xylose and dextrin,
organic acids such as gluconic acid, pyruvic acid and succinic
acid, amino acids such as alanine and serine and aromatic
hydrocarbons such as vanillin, ferulic acid and benzoic acid. Any
one of the above-listed substances or a combination of two or more
than two of them may be used as carbon source for the purpose of
the present invention.
[0049] There are no particular limitations to nitrogen sources to
be used for the purpose of the present invention so long as they
have utilization potential for microorganisms of the genus
Novosphingobium. Examples of nitrogen sources include organic
nitrogen sources such as peptone, polypeptone, bacto peptone, meat
extracts, yeast extracts, corn steep liquor, soybean flour, soybean
cake and yeast extracts and inorganic nitrogen sources such as
ammonium chloride, ammonium sulfate, urea, ammonium nitrate, sodium
nitrate and ammonium phosphate. Any one of the above listed
substances or a combination of two or more than two of them may be
used as nitrogen source for the purpose of the present
invention.
[0050] Examples of inorganic substances that can be used for the
purpose of the present invention include calcium salts, magnesium
salts, potassium salts, sodium salts, phosphates, manganese salts,
zinc salts, iron salts, copper salts, molybdenum salts and cobalt
salts. More specifically, examples of inorganic substances include
potassium dihydrogenphosphate, dipotassium hydrogenphosphate
magnesium sulfate, iron(I) sulfate, manganese sulfate, zinc
sulfate, sodium chloride, potassium chloride and calcium chloride.
Any one of the above listed substances or a combination of two or
more than two of them may be used for the purpose of the present
invention. Additionally, examples of micronutrients that can be
used for the purpose of the present invention include amino acids
and micronutrients vitamins such as biotin and thiamine.
[0051] There are no particular limitations to the concentrations of
lignins, lignin-related substances and culture medium ingredients
and also to the quantities of various ingredients such as the
number of microorganisms of the genus Novosphingobium in an action
system to be used for the purpose of the present invention. In
other words, such concentrations and quantities can appropriately
be selected. Additionally, it is preferable to agitate and shake
the action system in order to raise the frequency of contact
between lignins and lignin-related substances and microorganisms of
the genus Novosphingobium. There are no particular limitations to
the duration of any action in the action system so long as
production of one or more than one phenyl propane-based compounds
is recognized in the action system. Typical duration of an action
is between several hours and several days and the duration can
appropriately be selected as a function of the culturing method to
be adopted for the action system. When a batch culture method is
adopted, the duration of an action is preferably between about 12
hours and 36 hours or more than 36 hours.
[0052] The phenyl propane-based compound or compounds produced in
an action system can be used as they are without requiring any
particular treatment. However, the produced phenyl propane-based
compounds are preferably brought into a roughly refined state by
isolating them from microorganisms of the genus Novosphingobium
after the end of the action. Additionally, the produced phenyl
propane-based compounds can be refined by using a normally known
means for refining aromatic compounds such as a solid phase
extraction method or a chromatogram method. Furthermore, the phenyl
propane-based compounds can be obtained as solid by evaporating the
solvent and drying the compounds.
[0053] As far as the object of the present invention is achievable,
various steps and operations may be added to the production method
of the present invention before and/or after the above defined
steps.
[0054] Now, a specific exemplar mode of carrying out the production
method of the present invention will be described below.
[0055] Lignin-containing biomass is suspended in water or dioxane
and the suspension is kept warm at temperatures between 20 and
80.degree. C. in a water bath for a period between an hour and ten
hours. After the temperature keeping period, the solid components
are removed from the suspension to obtain solution of extracted
lignins. Then, an alkali compound is dropped to the lignin-extract
solution to a small extent to adjust the pH value and make the
solution weakly acidic. Subsequently, nutrient culture medium
ingredients and a magnesium salt are added to the lignin extract
solution. Then, the obtained solution is sterilized and
microorganisms of the genus Novosphingobium are inoculated into the
sterilized solution. After culturing the microorganisms at
temperatures between 15 and 40.degree. C. for a period between 10
and 120 hours, the obtained culture solution is subjected to a
centrifugal operation to obtain culture supernatant. The target
compounds, which are phenyl propane-based compounds, are refined
from the culture supernatant and then dried and solidified in inert
gas to obtain solid phenyl propane-based compounds.
[0056] The phenyl propane-based compounds obtained by the
production method of the present invention can be utilized as
starting materials or intermediates for producing resins, adhesive
agents, resist materials and drugs. More specifically, when
3-hydroxy-1-(4-hydroxy-3-methoxyphenyl)-1-propanone is obtained by
the production method of the present invention, the compound can be
transformed into coniferyl alcohol and so on that are industrially
useful as starting materials of drugs, perfumes, food materials and
so on by way of 1-(4-hydroxy-3-methoxyphenyl)-1,3-propanediol.
[0057] Microorganisms that can be used for the production method of
the present invention are those of Novosphingobium sp. MBES04
(accession number: NITE P-01797). MBES04 strain is expressed as
Novosphingobium sp. MBES04 for microorganism identification and was
deposited in the Fermentation Res. Inst. Microorganism Depository
Center (292-0818 2-5-8 Kazusakamatari, Kisarazu City, Chiba
Prefecture) of the National Institute of Technology and Evaluation
(NITE) on the deposition date of Jan. 30, 2014 with accession
number of "NITE P-01797". Additionally, Novosphingobium sp. MBES04
was transferred from national deposit to international deposit in
the NITE Patent Microorganism Depositary Center on the deposition
date of Feb. 9, 2015 (accession number: NITE ABP-01797).
[0058] As described above, microorganisms that can be used for the
purpose of the present invention can be isolated from
microorganisms existing in woods or the like where corrosion and
rottenness are observed, by using the mycological properties, the
physiological properties and the utilization potential as substrate
and/or the base sequence of MBESS04 strain as indexes.
[0059] While there are no particular limitations to the method of
culturing microorganisms of MBES04 strain, examples of culture
methods that can be used for the purpose of the present invention
include liquid culture methods (shaking culture method and aeration
and agitation culture method) and preferably an aerobic liquid
culture method is employed. From the industrial point of view, the
use of an aeration and agitation culture method is more preferable.
Any culture time may be used for the purpose of the present
invention so long as microorganisms of MBES04 strain starts growing
within the selected culture time. The culture time is, for example,
between 8 and 120 hours. There are no particular limitations to the
method of confirming the growth of microorganisms of MBES04 strain.
For example, a culture specimen may be taken and observed through a
microscope or observed for light absorption. Additionally, there
are no particular limitations to the dissolved oxygen concentration
of the culture solution but it is typically between 0.5 and 20 ppm.
For the purpose of maintaining aerobic culturing conditions, the
aeration rate needs to be regulated and the culture solution needs
to be agitated, while oxygen may need to be added to the air to be
used for aeration. Batch culture, fed-batch culture, continuous
culture or perfusion culture may be used as culturing method for
the purpose of the present invention.
[0060] Microorganisms to which any known microorganism utilization
technique is applicable can be used for the purpose of the present
invention. More specifically, microorganisms in various modes of
existence may be used. Such microorganisms include not only
microorganisms existing in liquid but also microorganisms in a
state of being adsorbed or buried in a carrier and microorganisms
in a freeze-dried state. Microorganisms that can be used for the
purpose of the present invention may be a component of a kit for
producing one or more than one phenyl propane-based compounds. When
biomass is packaged with a solvent suitable for extracting lignins
and one or more than one culture medium ingredients, it is possible
to determine if the obtained biomass can be used as starting
material for producing phenyl propane-based compounds.
[0061] Now, the present invention will be described in greater
detail below by way of examples. It should be noted, however, that
the present invention is by no means limited to the examples that
are described below and the present invention can be carried out in
various different modes so long as the problem to be solved by the
present invention can actually be solved in any of such modes.
EXAMPLES
Example 1
Screening of Lignin-Metabolizing Microorganisms
[0062] A mixture obtained by suspending 0.2% W/V of sawdust of
Japanese beech and 2% W/V of BACTO Agar in artificial seawater was
treated in an autoclave at 121.degree. C. for 15 minutes. After the
autoclave treatment, the mixture was cooled to 60.degree. C. and
then IMK culture medium (available from Daigo) was added according
to the procedure manual annexed to the product and supplied by the
manufacturer. After adding the IMK culture medium, the mixture was
solidified in a petri dish to turn it into a solid culture medium
for isolating microorganisms.
[0063] A sunken coniferous tree that was eaten away by shipworms
and well rotten was collected from the 260-m deep bottom of Suruga
Bay. A 0.1 g sample of the sunken tree was powdered and suspended
in artificial sea water (available from Daigo) and several drops of
the suspension were applied onto the solid culture medium for
isolating microorganisms. Then, the solid culture medium was held
warm at 25.degree. C. for 10 days. Thereafter, the microorganisms
that had formed colonies on the solid culture medium were
transferred onto Difco Marine Agar 2216 (available from Becton
Dickinson) and repeatedly subjected to streak culture at 25.degree.
C. to obtain about 1,000 strains of microorganisms of a single
genus.
[0064] A culture medium was prepared by mixing Tripticase Soy Broth
(available from Becton Dickinson) and Difco Marine Broth 2216
culture medium (available from Becton Dickinson) at a ratio of 1:1
and guaiacylglycerol-.beta.-guaiacyl ether (Compound I) was added
to the culture medium so as to obtain a final concentration of 1
mM. In this way, a Compound I-containing culture medium was
prepared. The isolated microorganisms were inoculated into the
Compound I-containing culture medium. After culturing the
microorganisms at 25.degree. C. and 120 rpm for 3 days, culture
supernatant was obtained by means of a centrifugal operation
conducted at 4,800 rpm for 5 minutes. 0.9 mL of methanol was added
to 0.1 mL of the culture supernatant and mixed. Then, the mixture
was subjected to a centrifugal operation conducted at 4,800 rpm for
5 minutes. The obtained culture supernatant was subjected to a
reverse phase HPLC analysis under the following conditions. A
decrease of Compound I was detected as a result of disappearance of
a peak around the retention time of 5.7 minutes on the basis of a
chromatogram showing absorbance at UV 270 nm.
Reverse Phase HPLC Conditions
[0065] Column; Xbridge OST C18 (available from WATERS), 4.6 mm
I.d..times.100 mm L. eluents; (A) [2 mM of ammonium acetate, 0.05%
V/V formic acid], (B) methanol; solution feeding rate; 0-1 minutes
10% V/V (B), 1-8 minutes 10% V/V(B)-90% V/V (B), column
temperature; 40.degree. C., flow rate: 1.2 ml/min, detection;
Photodiode Array Detector UV 200-500 nm (PDA model 2998, available
from WATERS)
[0066] As a result of a reverse phase chromatography analysis of
the culture supernatant, it was found that Compound I had
completely disappeared from the culture supernatant inoculated with
the microorganism strain named as MBES04 strain. As a result, it
was found that the MBES04 strain was that of microorganisms having
a potential ability of utilizing lignins and lignin-related
substances.
Example 2
Identification of Metabolites Due to Degradation of Compound I by
MBES04 Strain
[0067] A Compound I-containing culture medium was prepared by
adding Compound I to a medium produced by mixing Tripticase Soy
Broth (available from Becton Dickinson) and Difco Marine Broth 2216
culture medium (available from Becton Dickinson) at a ratio of 1:1
so as to obtain a final concentration of 3 mM of Compound I and
microorganisms of MBES04 strain were inoculated into the Compound
I-containing culture medium. Part of the culture solution was
extracted every 6 hours after the inoculation of microorganisms of
MBES04 strain and the culture supernatant obtained by means of a
centrifugal operation was subjected to a reverse phase HPLC
analysis each time. As a result, it was found that, as Compound I
decreases, new compounds are produced at retention time of about
4.4 minutes and at retention time of about 5.5 minutes respectively
(see FIG. 1). These compounds are respectively named as Compound
III and Compound VI. Compound VI was identified as guaiacol
(Guaiacol in FIG. 2) on the basis of the retention time and the
absorption spectrum thereof. It was confirmed that Compound III had
been accumulated in the culture medium without being subjected to
any additional metabolic transformation.
[0068] In order to identify Compound III, the above culture
supernatant was refined by means of a solid phase extraction method
(OASIS WAX; available from WATERS) and subjected to reverse phase
ultraperformance liquid chromatography (UPLC)-time-of-flight
precision mass spectrometry (ACCUITY UPLC H-Class, XevoG2 QTOF,
available form WATERS) under the following conditions.
Reverse Phase UPLC Conditions (UPLC-Time-of Flight Precision Mass
Spectrometry)
[0069] Column; ACQUITY UPLC BEH C18 Column, 130 angstroms, 1.7
.mu.m, (available from WATERS) , 2.1 mm I.d..times.100 mm L (Part
Number: 186002352).
eluent; (A) [2 mM of ammonium acetate, 0.05% V/V formic acid], (B)
[95% V/V acetonitrile], solution feeding rate; 0-5 minutes 5%
V/V-95% V/V(B), 5-7 minutes 95% V/V(B), column temperature;
40.degree. C. flow rate; 0.4 ml/min.
Mass Spectrometry Conditions (UPLC-time-of Flight Precision Mass
Spectrometry)
[0070] Detection mass range 100-1,000 Da, data acquisition scanning
interval 0.1 seconds, desolvation gas temperature 500.degree. C.,
ion source ESI negative mode ion source temperature 150.degree. C.,
cone voltage 20 V.
[0071] As a result of the reverse phase UPLC-time-of-flight
precision mass spectrometry, m/z195.1 ions were detected from
Compound III, and the molecular weight of Compound III was 196.1
and the compositional formula of Compound III was estimated to be
C.sub.10H.sub.12O.sub.4.
[0072] Substantially, Compound III was extracted three times from
the microorganism culture supernatant by means of 100 mL of ethyl
acetate. The ethyl acetate layer was condensed, dried and
solidified under reduced pressure to obtain 0.5 g of a crude
product. The obtained crude product was refined by column
chromatography in a manner as described below and 0.14 g of refined
crystal was obtained from the fraction containing Compound III by
evaporating the eluent solvent.
[0073] Column: silica gel (WAKO gel 200, available from WAKO Pure
Chemical Industries) 2.1 cm I.d..times.110 mm L Eluent: ethyl
acetate/toluene=2/3
[0074] Dispersed Compound III was dissolved in deuterochloroform
and subjected to a spectrum obtaining operation using a 400 MHz
nuclear magnetic resonance apparatus to obtain 1H-NMR, 13C-NMR
spectrum.
[0075] The chemical shift values obtained by an NMR analysis of
refined Compound III are listed below.
1H-NMR .delta. (ppm) : 3.19 (t, J=4.4, 2H, .beta.-H), 3.96 (s, 3H,
O--CH.sub.3), 4.02 (t, J=4.4, 2H, .gamma.-H), 6.12 (s, 1H,
phenol-OH), 6.97 (d, J=6.8 1H, Ar-H), 7.55(m, 2H, Ar-H). 13C-NMR
.delta. (ppm): 39.753 (.beta.-C), 56.086 (OCH.sub.3-C) , 58.333
(.gamma.-C) , 109.549, 113.927, 123.660, 129.658, 146.677, 150.744
(Ar-C), 199.096 (.alpha.-C).
[0076] From the results of the above described analyses, Compound
III was identified as
3-hydroxy-1-(4-hydroxy-3-methoxyphenyl)-1-propanone and it was
found that a compound having a phenyl propane structure had been
obtained.
Example 3
Identification of MBES04 Strain
[0077] A culture medium was prepared by mixing Tripticase Soy Broth
(available from Becton Dickinson) and Difco Marine Broth 2216
culture medium (available from Becton Dickinson) at a ratio of 1:1.
Then, microorganisms of MBES04 strain were cultured on the prepared
culture medium at 25.degree. C. and 120 rpm for 3 days and then
subjected to a centrifugal operation at 4,800 rpm for 5 minutes to
obtain cells of MBES04 strain. Then, all the DNAs were extracted
from the cells of MBES04 strain by means of the DNA extraction kit
NucleoSpin (registered trademark) Plant II (available from TAKARA
BIO). A PCR was made to take place by using all the DNAs as
template and also using primer 27f (5'-AGAGTTTGATCCTGGCTCAG-3')
(sequence number 1) and 1525r (5'-AAAGGAGGTGATCCAGCC-3') (sequence
number 2) and the base sequence of the obtained amplified fragment
was analyzed. The conditions used for the PCR and those used for
the base sequence analysis are as follow.
PCR Conditions
[0078] 1.times.PCR buffer (containing MgCl.sub.2) :200 .mu.m dNTPs,
0.6 .mu.m 27f, 0.6 .mu.m 1525r, 1.4 U of LA Taq DNA (Polymerase
available from TAKATA BIO) Thermal Cycler temperature condition
97.degree. C. 2 minutes, [97.degree. C. 30 seconds, 60.degree. C. 1
minute, 72.degree. C. 90 seconds].times.30 cycles, 72.degree. C. 5
minutes
Base Sequence Analysis
[0079] BigDye (registered trademark) Terminator v3.1 Cycle
Sequencing Kit, v3.1 (available from Applied Biosystems) ABI 3730
XL DNA Analyzer (available from Applied Biosystems)
[0080] A BLAST sequence retrieval operation was conducted on the
base sequence obtained by the above described base sequence
analysis (DNA base sequence for encoding 16S ribosomal RNA)
(sequence number 3) (Accession Number: AB733576) relative to NCBI
nucleotide database16S ribosomal RNA sequences (Bacteria and
Archaea) (http://www.ncbi.nlm.nih.gov/blast) by means of the
Megablast (Optimize for highly similar sequences) method to find
that microorganisms of MBES04 strain are those that belong to the
genus Novosphingobium.
[0081] Additionally, it was found that the species that are closest
to MBES04 strain are Novosphingobium pentaromativorans (Accession
Number NR_041046) and Novosphingobium panipatense (Accession Number
NR_044210). However, since the degree of agreement (identity) of
16S ribosomal RNA-DNA was 97%, microorganisms of MBES04 strain were
determined to be those of anew species belonging to the genus
Novosphingobium. Thus, the inventors of the present invention named
the MBES04 strain as Novosphingobium species MBES04 and a
conservation sample thereof was deposited in the Fermentation Res.
Inst. Microorganism Depository Center (292-0818 2-5-8
Kazusakamatari, Kisarazu City, Chiba Prefecture) of the National
Institute of Technology and Evaluation (NITE) on the deposition
date of Jan. 30, 2014 with identification name of "Novosphingobium
sp. MBES04" and accession number of "NITE P-01797".
Example 4
Investigation of Properties of MBES04 Strain
[0082] Microorganisms of MBES04 strain could be cultured under the
following conditions.
culture medium: liquid nourishment/solid culture medium containing
MgS.sub.04 by 0.1% W/V temperature: 10-45.degree. C. (optimum
temperature 30.degree. C.) pH: 5.5-8.5 (optimum pH6) NaCl
concentration: 0-6% W/V (optimum concentration 1% W/V)
[0083] The mycological properties of microorganisms of MBES04
strain are as follow.
(1) Morphological Property
[0084] bacillus of 0.3 to 0.8.times.2 to 3 .mu.m
(2) Cultural Properties
[0085] Microorganisms of MBES04 strain form circular colonies of a
diameter of between 0.5 and 1.5 mm on an agar plate culture medium
(0.1% W/V MgSO.sub.4-added LB culture medium); at 30.degree. C. in
48 hours. All the peripheral edges of the colonies were smooth,
lustrous and yellowy and showed a lowly convex profile.
[0086] As for the physiological properties of microorganisms of
MBES04 strain, the results obtained by a physiological property
test using an APIZYME are shown in Table 1 below. In Table 1, +
indicates an instance where enzyme activity was observed and -
indicates an instance where no enzyme activity was observed.
TABLE-US-00001 TABLE 1 enzyme activity alkaline phosphatase +
esterase (C4) - esterase lipase (C8) - lipase (C14) + leucine
arylamidase + valine arylamidase + cystine arylamidase + trypsin -
.alpha.-chymotrypsin - acid phosphatase +
naphthol-AS-BI-phosphohydrolase + .alpha.-galactosidase -
.beta.-galactosidase + .beta.-glucoronydase + .alpha.-glucosidase -
.beta.-glucosidase + N-acetyl-.beta.-glucosaminidase -
.alpha.-mannosidase - .alpha.-fucosidase -
[0087] Table 2 below shows the results obtained from a test on the
utilization potential ability of MBES04 strain as substrate by
using a GN2 plate (available from BIOLOG). In Table 2, + indicates
an instance where the ability of utilization is observed and +
indicates an instance where the ability of utilization is not
observed.
TABLE-US-00002 TABLE 2 .alpha.- .beta.- Cyclodextrin Cyclodextrin
Dextrin Glycogen Inulin Mannan (-) (+) (+) (-) (-) (-) L-
D-Arabitol Arbutin D- D- L-Fucose D- Arabinose (+) (-) Cellobiose
Fructose (+) Galactose (+) (+) (-) (-) .alpha.-D- Lactulose Maltose
Maltotriose D-Mannitol D- D- Lactose (+) (+) (+) (-) (-) Mannose
Melezitose (-) (-) .beta.- .alpha.- Palatinose D-Psicose D- L-
D-Ribose MethylD- MethylD- (-) (-) Raffinose Rhamnose (-) glucoside
mannoside (-) (-) (+) (+) D- D- Turanose Xylitol D-Xylose Acetic
acid .alpha.- Tagatose Trehalose (+) (+) (+) (-) Hydroxybutyric (-)
(-) acid (-) Lactamide D-Lactic L-Lactic D-Malic L-Malic Methyl
Mono- (+) acid methyl acid acid (+) acid pyruvate methyl ester (+)
(-) (-) (+) succinate (+) L- D-Alanine L-Alanine L-Alanyl- L-
L-Glutamic Glycyl-L- Alaninamide (+) (-) gycline Asparagine acid
(-) glutamic (-) (-) (-) Acid (+) Adenosine 2'- Inosine Thymidine
Uridine Adenosine- Thymidine- (-) Deoxyadenosine (-) (-) (-) 5'-
5'- (+) monophosphate monophosphate (+) (+) N-Acetyl- N-Acetyl- D-
D- Tween 40 Tween 80 Glucosamine Mannosamine Amygdalin (-) (-) (+)
(-) (+) L- D- Gentiobiose D-Gluconic .alpha.-D- m-Inositol
Arabinose Galacturonic (-) acid Glucose (+) (+) acid (+) (+) (-)
.alpha.-D- D- .alpha.- .beta.-Methyl 3- .alpha.-Methyl Lactose (+)
Melibiose MethylD- D- Methylglucose D- (+) galactoside galactoside
(+) glucoside (+) (-) (-) .beta.- Salcin Sedoheptulosan D-Sorbitol
Stachyose Sucrose MethylD- (-) (+) (-) (+) (-) glucoside (+) D-
.beta.- .gamma.- p- .alpha.-Keto .alpha.-Keto Tagatose
Hydroxybutyric Hydroxybutyric Hydroxyphenyl Glutaric valericacid
(-) acid acid (-) acetic acid (-) (+) acid (-) (-) Lactamide
Propionic Pyruvic Succinamide Succine N-Acetyl (+) acid (+) acid
acid acid (+) L-glutamic (+) (+) acid (+) L- L- L-Serine Putroscine
2,3- Glycerol Alaninamide Pyroglutamic (+) (+) Butanediol (-) (-)
acid (+) (-) Adenosine Uridine-5'- Fructose- Glucose- Glucose-
D-L-.alpha.- (-) monophosphate 6- 1- 6- Glycerolphosphate (-)
phosphate phosphate phosphate (+) (+) (+) (+)
[0088] The properties of Novosphingobium pentaromativorans and
those of Novosphingobium panipatense as described in Non-Patent
Literature "Internal J Syst Evol Microbial. 2009 January; 59 (Pt
1): 156-61 Gupta S K et al. (which is incorporated by reference
herein in its entirety) and those of MBES04 strain were compared.
Table 3 shows the results of the comparison. From the results of
the investigation on the properties of microorganisms of MBES04
strain, it was found that MBES04 strain is a strain of
microorganisms that is different from Novosphingobium
pentaromativorans and Novosphingobium panipatense in terms of that
MBES04 strain has at least a potential ability of utilizing
xylose.
TABLE-US-00003 TABLE 3 N. panipatense N. pentaromativorans MBES04
SM16.sup.T (*.sup.) DSM 17173.sup.T(*.sup.) Growth at/in: 4.degree.
C. - - + 41.degree. C. + ++ + 5% NaCl + - + Assimilation of:
Sucrose - (+) + Malonate + - (+) Mannose - + - Mannitol - - -
Arabinose + + + Fructose - + - Galactose - + - Rhamnose - - +
Trehalose - + + Xylose + - - Sorbitol - + - Glycerol - - - Serine +
+ - ++ strongly positive + positive (+) weekly positive - negative
.sup.(*.sup.) Data from Gupta S K et al. Int J Syst. Evol
Microbiol. January 2009; 59(Pt 1): 156-61.
Example 5
Production of Compounds Having a Phenyl Propane Structure from
Glass Plant-Based Biomass
[0089] Compounds having a phenyl propane structure were produced
from extract solution of rice straws by following the procedures
described below.
[0090] 50 g of dried and powdered rice straw was suspended in a
mixture solution of 300 mL of dioxane, which is widely recognized
as solvent suitable for extracting lignins, and 15 mL of water and
left in an immersed state at room temperature for 6 days. After the
immersion treatment, the suspension was subjected to an operation
of filtering the solid content of the suspension by means of filter
paper to obtain a filtrate. After drying and solidifying the
filtrate under reduced pressure by means of an evaporator, the
evaporation residue was dried by means of a desiccator to obtain
2.65 g of solid. Then, the solid was dissolved by adding 50 ml of
ethyl acetate and 70 mL of pure water to the solid. Thereafter, the
ethyl acetate layer was extracted and dried under reduced pressure
by means of an evaporator to obtain 0.86 g of solid. The solid was
then dissolved in DMF so as to make it show a concentration of 10%
W/V to obtain lignin-containing rice straw extract solution.
[0091] Difco Yeast Extract (available from Becton Dickinson) and
MgS.sub.04 (available from WAKO Pure Chemical Industries) were
added to ion exchange water so as to make them respectively show
final concentrations of 0.5% W/V and 0.1% W/V and subsequently a
small amount of 1N NaOH was dropped in the solution so as to make
the solution show a pH value of 6.5. Then, the solution was
filtered and sterilized by means of a 0.22 .mu.M filter. The above
described lignin-containing rice straw extract solution was
aseptically added to the obtained filtrate by 1/40 capacity to
produce an aseptic culture medium. The dioxin extract concentration
in the aseptic culture medium was 2.5 mg/mL. The aseptic culture
medium was subjected to a reverse phase UPLC-time-of-flight
precision mass spectrometry under the conditions described in
Example 2 but no guaiacylglycerol-.beta.-guaiacyl ether (Compound
I) was detected.
[0092] Microorganisms of MBES04 strain were inoculated into the
obtained aseptic culture medium and cultured by shaking the culture
medium at 30.degree. C. for 48 hours. Subsequently, the culture
solution was subjected to a centrifugal operation at 10,000 rpm for
5 minutes to obtain culture supernatant. 0.5 mL of the obtained
culture supernatant was refined by means of a solid phase
extraction method (OASIS WAX; available from WATERS) and then dried
and solidified in nitrogen gas. After the drying and solidifying
process, the residue was dissolved in 0.5 mL of 20% V/V
acetonitrile and subjected to reverse phase UPLC-time-of-flight
precision mass spectrometry under the conditions described in
Example 2. As a control, a sample culture medium that had not been
inoculated with microorganisms of MBES04 strain was processed in
the same way and analyzed.
[0093] As a result of the reverse phase UPLC-time-of-flight
precision mass spectrometry, it was confirmed that Compound III had
been produced by 1.10 .mu.g/mL due to the action exerted by the
microorganisms of MBES04 strain. In the instance where no
microorganisms of MBES04 strain had been inoculated, the comparable
figure was 0.02 .mu.g/mL. The yield was 0.04% W/W relative to the
fractionated dry solid that is soluble to rice straw-containing
dioxane ethyl acetate. As a result of the analysis, ions (225.1
m/z) of a compound having a molecular weight greater than that of
Compound III by about 30 were detected. It corresponds to a
compound having an additional methoxy group if compared with
Compound III. By calculations on the basis of the calibration curve
(without sensitivity correction) of
3-hydroxy-1-(4-hydroxy-3-methoxyphenyl)-1-propanone, the
concentration of the compound was determined to correspond to 0.08
.mu.g/mL from the peak area value of the mass chromatogram of the
compound. Additionally, ions (165.1 m/z) of a compound having a
molecular weight smaller than that of Compound III by about 30 were
also detected. It corresponds to a compound that is short of a
methoxy group if compared with Compound III. By calculations on the
basis of the calibration curve (without sensitivity correction) of
3-hydroxy-1-(4-hydroxy-3-methoxyphenyl)-1-propanone, the
concentration of the compound was determined to correspond to 0.73
.mu.g/mL from the peak area value of the mass chromatogram of the
compound. In the case of the sample where no microorganisms of
MBES04 strain were inoculated, the concentration of Compound III
and its derivatives was not greater than 0.01 .mu.g/mL.
Example 6
Production of Compounds Having a Phenyl Propane Structure from
Wood-Based Biomass
[0094] Compounds having a phenyl propane structure were produced
from oak sawdust extract solution by following the procedures
described below.
[0095] 20 g of dried and powdered oak sawdust was suspended in a
mixture solution of 100 mL of dioxane and 5 mL of water and held in
an immersed state at room temperature for 5 days. After the
immersion, the solid fraction was removed by filtering the solution
by means of filter paper to obtain a filtrate.
[0096] The obtained filtrate was dried by means of an evaporator
under reduced pressure to obtain 0.53 g of an oak dioxane extract.
The oak dioxane extract was then dissolved in 10% W/V of DMF. Difco
Yeast Extract (available from Becton Dickinson) and MgSO.sub.4
(available from WAKO Pure Chemical Industries) were added to ion
exchange water so as to make them respectively show final
concentrations of 0.5% W/V and 0.1% W/V and subsequently a small
amount of 1N NaOH was dropped in the solution so as to make the
solution show a pH value of 6.5. Then, the solution was filtered
and sterilized by means of a 0.22 .mu.M filter. The DMF solution of
the above-described oak dioxane extract was aseptically added to
the obtained filtrate by 1/40 capacity to produce an aseptic
culture medium. The oak dioxane extract concentration in the
aseptic culture medium was 2.5 mg/mL. The aseptic culture medium
was subjected to reverse phase UPLC-time-of-flight precision mass
spectrometry under the above described conditions but no
guaiacylglycerol-.beta.-guaiacyl ether (Compound I) was
detected.
[0097] Microorganisms of MBES04 strain were inoculated into the
obtained aseptic culture medium and cultured by shaking the culture
medium at 30.degree. C. for 48 hours. Subsequently, the culture
solution was subjected to a centrifugal operation at 10,000 rpm for
5 minutes to obtain culture supernatant. 0.5 mL of the obtained
culture supernatant was refined by means of a solid phase
extraction method (OASIS WAX; available from WATERS) and then dried
and solidified in nitrogen gas. After the drying and solidifying
process, the residue was dissolved in 0.5 mL of 20% V/V
acetonitrile and then subjected to reverse phase
UPLC-time-of-flight precision mass spectrometry under the above
described conditions. As a control, a sample culture medium that
had not been inoculated with microorganisms of MBES04 strain was
processed in the same way and analyzed.
[0098] As a result of the reverse phase UPLC-time-of-flight
precision mass spectrometry, it was confirmed that Compound III had
been produced by 0.93 .mu.g/mL due to the action exerted by the
microorganisms of MBES04 strain. In the instance where no
microorganisms of MBES04 strain had been inoculated, the comparable
figure was 0.02 .mu.g/mL. The yield was 0.04% W/W relative to the
oak dioxane extract. As a result of the analysis, ions (225.1 m/z)
of a compound having a molecular weight greater than that of
Compound III by about 30 were detected. It corresponds to a
compound having an additional methoxy group if compared with
Compound III. By calculations, the concentration of the compound
was determined to correspond to 0.30 .mu.g/mL from the peak area
value of the mass chromatogram of the compound. Additionally, as a
result of the reverse phase UPLC-time-of-flight precision mass
spectrometry, ions (165.1 m/z of a compound having a molecular
weight smaller than that of Compound III by about 30 were also
detected due to the action exerted by the microorganisms of MBES04
strain. By calculations on the basis of the calibration curve
(without sensitivity correction) of
3-hydroxy-1-(4-hydroxy-3-methoxyphenyl)-1-propanone, it was
confirmed that the compound had been produced to a concentration
corresponding to 0.90 .mu.g/mL from the peak area value of the mass
chromatogram of the compound.
Example 7
Production of Compounds Having a Phenyl Propane Structure from
Agricultural Waste-Based Biomass
[0099] Compounds having a phenyl propane structure were produced
from a waste Shiitake mushroom bed by following the procedures
described below.
[0100] 10 g of a dried and powdered waste Shiitake mushroom bed was
suspended in ion exchange water so as to make it show a
concentration of 10% W/V and held in a warmed condition at
60.degree. C. in a water bath for 3 hours. After the end of the
warmed condition, the solid fraction was removed from the
suspension by filtering using filter paper to obtain a filtrate.
The weight of the dried and removed solid was 1.88 g. As a result
of subjecting the filtrate to UPLC-time-of-flight precision mass
spectrometry, guaiacylglycerol-.beta.-guaiacyl ether (Compound I)
was not detected from the filtrate.
[0101] 1N NaOH was dropped into the obtained filtrate by a small
amount so as to adjust the pH of the filtrate to 6.5 and
subsequently Difco Yeast Extract (available from Becton Dickinson)
and then MgSO.sub.4 (available from WAKO Pure Chemical Industries)
were added to the filtrate so as to make them respectively show
final concentrations of 0.5% W/V and 0.1% W/V. The obtained
solution was filtered and sterilized by means of a 0.22 .mu.M
filter to produce an aseptic culture medium. Then, microorganisms
of MBES04 strain were inoculated into the aseptic culture medium.
After culturing the microorganisms at 30.degree. C. for 72 hours,
the culture solution was subjected to a centrifugal operation at
10,000 rpm for 5 minutes to obtain culture supernatant. Then, 0.5
mL of the obtained culture supernatant was refined by means of a
solid phase extraction method (OASIS WAX; available from WATERS)
and then dried and solidified in nitrogen gas. After the drying and
solidifying process, the residue of the drying and solidifying
process was dissolved in 0.5 mL of 20% V/V acetonitrile and
subjected to reverse phase UPLC-time-of-flight precision mass
spectrometry under the conditions described in Example 2.
[0102] As a result of the reverse phase UPLC-time-of-flight
precision mass spectrometry, it was confirmed that Compound III had
been produced by 2.96 .mu.g/mL due to the action exerted by the
microorganisms of MBES04 strain. In an instance where no
microorganisms of MBES04 strain had been inoculated, the comparable
figure was 0.09 .mu.g/mL. The yield was 0.02% W/W relative to the
hot water extract of the waste Shiitake mushroom bed. As a result
of the analysis, ions (225.1 m/z) of a compound having a molecular
weight greater than that of Compound III by about 30 were detected.
By calculations on the basis of the calibration curve (without
sensitivity correction) of
3-hydroxy-1-(4-hydroxy-3-methoxyphenyl)-1-propanone, the
concentration of the produced compound was determined to correspond
to 1.61 .mu.g/mL from the peak area value of the mass chromatogram
of the compound. Additionally, it was confirmed that ions (165.1
m/z) of a compound having a molecular weight smaller than that of
Compound III by about 30 had been produced. By calculations, it was
confirmed that the compound had been produced to a concentration
corresponding to 5.89 .mu.g/mL.
Example 8
Measurement of Production Efficiency of 3-hydroxy-1-
(4-hydroxy-3-methoxyphenyl) -1-propanone by Means of Microorganisms
of MBES04 Strain
[0103] Microorganisms of MBES04 strain were inoculated into 120 mL
of a culture medium containing 1M of
guaiacylglycerol-.beta.-guaiacyl ether (Compound I, to be
abbreviated as GGGE hereinafter) and 5 mM of MgSO.sub.4 LB culture
medium and cultured by shaking the culture medium at 30.degree. C.
2 mL of the culture solution was extracted every 6 hours from the
start of the culturing process to 48 hours after the start and
every 24 hours from 48 hours after the start, and subjected to a
centrifugal operation at 14,000 rpm for 5 minutes to obtain culture
supernatant. Then, 9-hydroxyfluoren was added to the culture
supernatant as internal standard material so as to make it show a
final concentration of 0.025 mM and the supernatant was diluted to
10-fold dilution by methanol. Then, the diluted supernatant was
subjected to a centrifugal operation at 14,000 rpm for 5 minutes to
remove the impurities. The obtained supernatant was then subjected
to an HPLC analysis under the conditions same as those described in
Example 1 and the decrease of Compound I and the newly produced
metabolite were quantified. The values obtained by the
quantifications were corrected on the basis of the peak area value
of the internal standard material in order to eliminate the
influence of the errors, if any, that had been involved in the
analysis process. The production ratio was determined by
computations by referring to the ratio when each of the components
was collected with the concentration same as the concentration at
the time of initial addition of 1 mM, which was defined as 100%. As
a result, it was found that Compound III could be collected with a
yield of not less than 50% after the elapse of 30 hours. Even when
the culture time was extended to 120 hours, Compound III did not
decrease and could be collected from the culture solution of MBES04
strain at a yield not less than 50% (see FIG. 1). FIG. 2 shows the
chromatogram obtained by using the samples obtained in 48 hours
from the start.
TABLE-US-00004 Primer 27f [Sequence Number 1] AGAGTTTGATCCTGGCTCAG
Primer 1525r [Sequence Number 2] AAAGGAGGTGATCCAGCC 16S ribosomal
RNA-DNA of MBES04 strain [Sequence Number 3]
AACGAACGCTGGCGGCATGCCTAACACATGCAAGTCGAACGAACC
CTTCGGGGTTAGTGGCGCACGGGTGCGTAACACGTGGGAATCTGC
CTCTTGGTTCGGAATAACAGTGAGAAATTACTGCTAATACCGGAT
GATGACTTCGGTCCAAAGATTTATCGCCAAGAGATGAGCCCGCGC
AGGATTAGGTAGTTGGTGGGGTAAAGGCCTACCAAGCCGACGATC
CTTAGCTGGTCTGAGAGGATGATCAGCCACACTGGGACTGAGACA
CGGCCCAGACTCCTACGGGAGGCAGCAGTGGGGAATATTGGACAA
TGGGCGAAAGCCTGATCCAGCAATGCCGCGTGAGTGATGAAGGCC
TTAGGGTTGTAAAGCTCTTTTACCAGGGATGATAATGACAGTACC
TGGAGAATAAGCTCCGGCTAACTCCGTGCCAGCAGCCGCGGTAAT
ACGGAGGGAGCTAGCGTTGTTCGGAATTACTGGGCGTAAAGCGCG
CGTAGGCGGTTACTCAAGTCAGAGGTGAAAGCCCGGGGCTCAACC
CCGGAACTGCCTTTGAAACTAGGTGACTAGAATCTTGGAGAGGTC
AGTGGAATTCCGAGTGTAGAGGTGAAATTCGTAGATATTCGGAAG
AACACCAGTGGCGAAGGCGACTGACTGGACAAGTATTGACGCTGA
GGTGCGAAAGTGTGGGGAGCAAACAGGATTAGATACCCTGGTAGT
CCACACCGTAAACGATGATAACTAGCTGTCCGGGTTCTTGGAATT
TGGGTGGCGCAGCTAACGCATTAAGTTATCCGCCTGGGGAGTACG
GTCGCAAGATTAAAACTCAAAGGAATTGACGGGGGCCTGCACAAG
CGGTGGAGCATGTGGTTTAATTCGAAGCAACGCGCAGAACCTTAC
CAGCGTTTGACATCCTCATCGCGATTTCCAGAGATGGATTTCTTC
AGTTCGGCTGGATGAGTGACAGGTGCTGCATGGCTGTCGTCAGCT
CGTGTCGTGAGATGTTGGGTTAAGTCCCGCAACGAGCGCAACCCT
CATCCTTAGTTGCCAGCATTTAGTTGGGCACTCTAAGGAAACTGC
CGGTGATAAGCCGGAGGAAGGTGGGGATGACGTCAAGTCCTCATG
GCCCTTACACGCTGGGCTACACACGTGCTACAATGGCGGTGACAG
TGGGCAGCAAGTGCGCGAGCACAAGCTAATCTCCAAAAGCCGTCT
CAGTTCGGATTGTTCTCTGCAACTCGAGAGCATGAAGGCGGAATC
GCTAGTAATCGCGGATCAGCATGCCGCGGTGAATACGTTCCCAGG
CCTTGTACACACCGCCCGTCACACCATGGGAGTTGGTTTCACCCG
AAGGTAGTGTGCTAACCGCAAGGAGGCAGCTAACCACGGTGGGAT
CAGCGACTGGGGTGAAGTCGTAACAAGGTAGCCGTAGGGGAACCT GC
INDUSTRIAL APPLICABILITY
[0104] 3-hydroxy-1-(4-hydroxy-3-methoxyphenyl)-1-propanone can be
obtained from biomass containing natural lignins and lignin-related
substances by means of the production method and microorganisms of
the present invention.
3-hydroxy-1-(4-hydroxy-3-methoxyphenyl)-1-propanone can be
transformed into various industrially useful compounds and also can
be used as starting material for producing
1-(4-hydroxy-3-methoxyphenyl)-1,3-propanediol and so on, which can
be utilized as starting material for producing resins, adhesive
agents, resist materials and drugs among others.
SEQUENCE LISTING
[0105] 15DF0302PCT_ST25.txt
Sequence CWU 1
1
3120DNAArtificial sequencePrimer 1agagtttgat cctggctcag
20218DNAArtificial sequencePrimer 2aaaggaggtg atccagcc
1831442DNANovosphingobium sp. MBES04 3aacgaacgct ggcggcatgc
ctaacacatg caagtcgaac gaacccttcg gggttagtgg 60cgcacgggtg cgtaacacgt
gggaatctgc ctcttggttc ggaataacag tgagaaatta 120ctgctaatac
cggatgatga cttcggtcca aagatttatc gccaagagat gagcccgcgc
180aggattaggt agttggtggg gtaaaggcct accaagccga cgatccttag
ctggtctgag 240aggatgatca gccacactgg gactgagaca cggcccagac
tcctacggga ggcagcagtg 300gggaatattg gacaatgggc gaaagcctga
tccagcaatg ccgcgtgagt gatgaaggcc 360ttagggttgt aaagctcttt
taccagggat gataatgaca gtacctggag aataagctcc 420ggctaactcc
gtgccagcag ccgcggtaat acggagggag ctagcgttgt tcggaattac
480tgggcgtaaa gcgcgcgtag gcggttactc aagtcagagg tgaaagcccg
gggctcaacc 540ccggaactgc ctttgaaact aggtgactag aatcttggag
aggtcagtgg aattccgagt 600gtagaggtga aattcgtaga tattcggaag
aacaccagtg gcgaaggcga ctgactggac 660aagtattgac gctgaggtgc
gaaagtgtgg ggagcaaaca ggattagata ccctggtagt 720ccacaccgta
aacgatgata actagctgtc cgggttcttg gaatttgggt ggcgcagcta
780acgcattaag ttatccgcct ggggagtacg gtcgcaagat taaaactcaa
aggaattgac 840gggggcctgc acaagcggtg gagcatgtgg tttaattcga
agcaacgcgc agaaccttac 900cagcgtttga catcctcatc gcgatttcca
gagatggatt tcttcagttc ggctggatga 960gtgacaggtg ctgcatggct
gtcgtcagct cgtgtcgtga gatgttgggt taagtcccgc 1020aacgagcgca
accctcatcc ttagttgcca gcatttagtt gggcactcta aggaaactgc
1080cggtgataag ccggaggaag gtggggatga cgtcaagtcc tcatggccct
tacacgctgg 1140gctacacacg tgctacaatg gcggtgacag tgggcagcaa
gtgcgcgagc acaagctaat 1200ctccaaaagc cgtctcagtt cggattgttc
tctgcaactc gagagcatga aggcggaatc 1260gctagtaatc gcggatcagc
atgccgcggt gaatacgttc ccaggccttg tacacaccgc 1320ccgtcacacc
atgggagttg gtttcacccg aaggtagtgt gctaaccgca aggaggcagc
1380taaccacggt gggatcagcg actggggtga agtcgtaaca aggtagccgt
aggggaacct 1440gc 1442
* * * * *
References